Since their introduction to the marketplace, Electronic Nicotine Delivery Systems (ENDS) have experienced a rapid growth in popularity as a means to deliver nicotine to the user in unprecedented high concentrations and purity. There is little evidence pertaining to the consequences this route of nicotine self- administration has on the health of the nave user or those who use this product to aid in cigarette smoking (CS) cessation. This Merit application is designed to fill gaps in our understanding of these issues. Here the focus will be on how the lung morphology and normal gene expression including a defined response to the inhaled house dust mite allergen is impacted by ENDS:nic exposure, especially in the lung previously exposed to cigarette smoke. There are 3 interactive Aims to address the Project Hypothesis: The use of nicotine as delivered by electronic nicotine delivery systems (ENDS:nic), both in the nave lung and subsequent to cessation of cigarette smoking (CS), impacts the morphology and immune responsiveness of the exposed lung and complicates normal recovery processes following CS cessation. Specific Aim 1. Does ENDS:nic exposure impact lung morphology, mucin production and fibrosis in the nave lung and the lung previously damaged by CS exposure? Measurement of ENDS:nic effects on the lung will include morphologic changes in alveolar spaces, mucin production (e.g., Muc5b) and deposition, and changes to collagen deposition (fibrosis). Results will determine how the form of nicotine (salt versus free-base) and its relative concentrations impact these parameters. We further hypothesize that ENDS:nic exposure (and possibly carrier alone) will alter lung morphology and that mice previously exposed to CS will not recover to the same extent as mice that are allowed to recover in the absence of END:nic use and possibly carrier. Specific Aim 2. Does ENDS:nic impact immune function in nave mice and does the transition from cigarette smoke to ENDS:nic exposure impede immune functional recovery? ENDS:Nic delivery strongly suppresses the lung eosinophil response to inhalation of the common house dust mite (HDM) allergen. This reliable experimental measurement provides an end-point to quantify the impact by different ionic forms of nicotine, their concentration and the associated e- liquid carrier compounds. The focus will also be on transcriptional responsiveness to HDM by AM and specific lung cellular signaling responses during recovery from prior CS-associated damage in the context of ENDS:nic relative to no product use as well as the impact by ENDS:nic itself. Specific Aim 3. How are alpha7-modulated cell-signaling mechanisms in the alveolar macrophage (AM) affected by ENDS:nic exposure? These experiments will focus on alveolar macrophages (AM) which constitute the majority of immune cells in the bronchial alveolar lung fluids (BALF). Newly developed methods that combine AM enrichment from the BALF of ENDS:nic and CS exposed mice and in vitro response to treatment with defined cytokines (IL-4+IL-10) will be done in the presence of specific inhibitors of cell signaling intermediates and positive allosteric modulators of the AM target of nicotine, the nicotinic receptor alpha7. The AM transcriptional response mechanisms will be measured using RT-PCR as well as RNA-Seq. Collectively, this study will provide the first comprehensive view of AM responsiveness after ENDS:nic exposure and how ENDS:nic use impacts the lung during recovery from previous cigarette smoking.